Target cell killing by cytotoxic T lymphocytes (CTLs) is critical for the immune response to viruses and tumors, is involved in transplant rejection and contributes to autoimmune disease pathogenesis. A key mechanism CTLs use to kill target cells is secretion of cell-killing agents from specialized lysosomal lytic granules. Screening a small molecule library for compounds that inhibit lytic granule exocytosis would 1) serve as an assay for blockers of the known signals that control the process; 2) reveal the presence of and define chemical probes for as-yet-unknown signals and 3) lead to novel immunomodulatory drugs that could enhance our ability to transplant organs and stem cells and to treat autoimmune disorders. CTL granule exocytosis can be monitored using fluorescently-labeled antibodies to detect the externalization of lysosome-associated membrane protein 1 (LAMP-1) from lytic granules to the plasma membrane in a simple no-wash assay protocol suitable for flow cytometry. We plan to capitalize on this simple assay together with the unique HTS flow cytometry capabilities at the University of New Mexico Center for Molecular Discovery (UNMCMD) to establish a phenotypic screen and to discover chemical probes that inhibit granulocyte exocytosis. We propose three specific aims. In the first, we will optimize assay conditions, determine DMSO tolerance, and apply repeated measures tests to assess assay robustness. We will measure in a low-throughput format dose-response curves for known inhibitors of different signaling pathways involved in granule exocytosis, then determine whether and at what concentrations these inhibitors can reliably be detected in a mock small-scale trial of the assay. In our second aim, also to be conducted in Year 1, we will transfer the assay to the UNMCMD, perform the validation steps needed to confirm assay performance in a high-throughput format, then screen the Prestwick Library of pharmaceuticals, a 7-plate validation set from the Molecular Libraries Small Molecule Repository (MLSMR) and the ICCB library library of 480 known bioactive compounds. We believe that completion of this aim by itself could substantially advance our knowledge of the signals controlling granule exocytosis. Finally, in year 2 (and of course contingent on success in aims 1 and 2) we will conduct the full screen of compounds in the MLSMR at the UNMCMD. PUBLIC HEALTH RELEVANCE: Cytotoxic T cell lytic granule exocytosis is important for killing virus infected cells, tumor cells and transplants, and is also involved in the etiology of some autoimmune disorders. Identifying small molecule inhibitors of granule exocytosis via high-throughput screening could lead to new clinically-important drugs that could be used as immunosuppressive agents, and could also provide important insights into the signaling pathways that control the process. This application is designed to determine whether a simple and robust high-throughput assay of lytic granule exocytosis based on the externalization of lysosome-associated membrane protein can be developed. If it can, we will use it to screen a large scale compound library for inhibitory compounds.